With the development of rapid, accurate, high-throughput autoantibody and potentially HLA assay systems, we are rapidly achieving the ability to screen and detect pre-diabetic subjects in the general population. Intervention studies in the NOD mouse suggest that antigen specific therapy can be effective in preventing diabetes. However, our current knowledge of the cellular response in human type 1 diabetes has not yet progressed enough to define relevant antigens to use in a similar fashion. The overall of this program project is to identify and assess peptide- specific immunomodulation strategies suitable for intervention therapy in patients with new-onset type 1 diabetes and pre-clinical islet autoimmunity. It rests on the hypothesis that by utilizing a novel approach of determining peptide immunogenicity in human HLA-transgenic mice bearing alleles associated with type 1 diabetes, we can establish the dominant epitopes of pre pro-insulin, GAD65 and ICA512 for a particular HLA allele. This analysis has been used by several groups including Project 1 to identify novel peptides that appear to be reactive in human type 1 subjects. This particular project will attempt to validate the analysis of peptide immunogenicity in HLA-transgenic mice (Project 1) by testing recognition of these peptides in HLA-defined new onset diabetic subjects and other unique pre-diabetic populations. We will test the hypothesis that identification of peptide specific responses is dependent on the HLA peptide interaction (identified in Project 1 and quantitated in Project 4). We will attempt to define this reactivity in proliferation and cytokine assays performed on selected antigen and peptide-specific T cell lines. We will extend these initial observations to include development of stable T cell lines and clones which can then be used to define the TCR alpha and beta chain usage of these antigen specific cells. Efforts will also be directed at obtaining a greater understanding of the immunologic phenotype associated with disease development by analyzing the differences in cellular and humoral immunity between individuals with high risk and protective HLA phenotypes identified by the genetics core (Project 6). This information will be important in defining hard immunological endpoints for potential future intervention trials. Additionally, we will determine the optimum dose, timing and frequency of administration of peptide in vaccination protocols in a animal model of type 1 diabetes. This type of information will be critically important to design to implement future clinical trials of peptide immunotherapy in human subjects. Together with our collaborators we hope to identify peptides of islet autoantigens which are immunodominant and recognized in human type 1 diabetes populations which in native or modified form might eventually form the basis of a trial of immunotherapy for this disease.